Abstract

Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease. The incidence of NAFLD has increased steadily due to its close association with the global epidemic of obesity and type 2 diabetes. However, there is no effective pharmacological therapy approved for NAFLD. Farnesoid X receptor (FXR), a member of the nuclear receptor subfamily, plays important roles in maintaining the homeostasis of bile acids, glucose, and lipids. FXR agonists have shown promise for the treatment of NAFLD. In this study, we report altenusin (2076A), a natural nonsteroidal fungal metabolite, as a novel selective agonist of FXR with an EC50 value of 3.2 ± 0.2 μM. Administration of 2076A protected mice from high-fat diet (HFD)-induced obesity by reducing the body weight and fat mass by 22.9% and 50.0%, respectively. Administration of 2076A also decreased the blood glucose level from 178.3 ± 12.4 mg/dl to 116.2 ± 4.1 mg/dl and the serum insulin level from 1.4 ± 0.6 ng/dl to 0.4 ± 0.1 ng/dl. Moreover, 2076A treatment nearly reversed HFD-induced hepatic lipid droplet accumulation and macrovesicular steatosis. These metabolic effects were abolished in FXR knockout mice. Mechanistically, the metabolic benefits of 2076A might have been accounted for by the increased insulin sensitivity and suppression of genes that are involved in hepatic gluconeogenesis and lipogenesis. In summary, we have uncovered a new class of nonsteroidal FXR agonist that shows promise in treating NAFLD and the associated metabolic syndrome.

The 2076A is a selective FXR agonist. (A) Chemical structure of 2076A. (B) Activation of the GAL4-hFXR LBD chimeric receptor by 2076A. Cells were cotransfected with GAL4-hFXR LBD and the GAL4-responisve luciferase report tk-UAS-Luc. The transfected cells were treated with increasing concentrations of 2076A for 24 hours before luciferase assay. (C) The 2076A did not activate GAL4 fusions of 14 other nuclear receptors. (D) Activation of the full-length FXR by 2076A. Cells were cotransfected with FXR and the FXR-responsive tk-EcRE-Luc luciferase report gene. The transfected cells were treated with increasing concentrations of 2076A for 24 hours before luciferase assay. (E) Lack of activation of CAR by 2076A. Cells were cotransfected with the full-length mouse CAR and the CAR-responsive tk-PBRE-Luc luciferase report. The transfected cells were treated with 2076A or TCPOBOP for 24 hours before luciferase assay. (F) The expression of FXR-responsive genes in primary hepatocytes isolated from WT and FXR KO mice. The hepatocytes were treated with vehicle or 2076A (10, 25, or 50 μM) for 24 hours before cell harvesting and real-time PCR analysis; CDCA (50 μM) was included as the positive control. The data represent mean ± S.D. n = 3 for each group. *P < 0.05; **P < 0.01 compared with the vehicle-treated group. The transfection results represent one of three independent experiments.

Computational docking analysis of the binding of 2076A to FXR. The docking analysis was based on the crystal structures of human FXR from the Protein Data Bank (PDB identification: 1OSH). The hydrogen bond interactions between 2076A and FXR are shown in yellow dash lines.

Treatment with 2076A inhibits HFD-induced obesity. WT (n = 4 for each group) and FXR KO mice (n = 5 for each group) were fed with HFD for 16 weeks and then treated i.p. with 2076A (30 mg/kg per day) for 3 weeks. (A) Representative appearance of mice at the end of the experiment. (B) Representative appearance of visceral of mice. (C) Body weight and body composition analysis. (D) Histologic analysis of the visceral fat by H&E staining. (E–G) The mRNA expression of mitochondrial uncoupling proteins UCPs in the white adipose tissue and skeletal muscle (E), adipocyte differentiation marker genes (F), and lipogenic genes (G) in the visceral fat was measured by real-time PCR. *P < 0.05; ***P < 0.001, compared with the vehicle-treated group. Note only the differences between vehicle group and 2076A-treated group within the same genotype (WT or FXR knockout) were compared, so the unpaired two-tailed Student’s t test was used.

Treatment with 2076A reverses HFD-induced hepatic steatosis. Mice were the same as described in . (A) Appearance of the livers at the end of the experiment. (B) Histologic analysis of liver sections by H&E staining. (C) Liver weight (left) and the ratio of liver weight to body weight (right). (D and E) The hepatic levels of triglycerides (D) and cholesterol (E). (F) The hepatic mRNA expression of genes involved in lipogenesis was measured by real-time PCR. WT, n = 4 for each group; FXR KO, n = 5 for each group. *P < 0.05; **P < 0.01, compared with the vehicle-treated group.

Treatment with 2076A sensitizes insulin signaling and inhibits hepatic gluconeogenesis in vitro and in vivo. (A) Treatment of primary hepatocytes with 2076A increased the basal phosphorylation of Akt (p-AKT), as shown by Western blotting. Shown on the right is the densitometric quantification of the blots. (B) Treatment of primary hepatocytes with 2076A elevated the insulin-stimulated phosphorylation of Akt (p-AKT), as shown by Western blotting. Shown on the right is the densitometric quantification of the blots. (C) WT mice were treated with vehicle or 2076A (30 mg/kg per day) for 1 week before tissue harvesting and measurement of p-AKT in the liver, white adipose tissue, and skeletal muscle. Shown on the right is the densitometric quantification of the blots. (D) The inhibitory effect of 2076A on the forskolin-stimulated glucose production in primary hepatocytes isolated from WT mice. (E) The effect of 2076A on the hepatic expression of gluconeogenic genes in WT (n = 4) and FXR KO (n = 5) mice. *P < 0.05; **P < 0.01, compared with the vehicle-treated group.